RNA sequences generated using a microarray having a base cleavable succinate linker

Abstract

There is disclosed a microarray having base cleavable succinate linkers. The microarray has a solid surface with known locations, each having reactive hydroxyl groups. The density of the known locations is greater than approximately 100 locations per square centimeter. Amino moieties are attached to the reactive hydroxyl groups. Preferably the attachment is through a phosphorous-oxygen bond between the phosphorous of amino amidite moieties and the oxygen of the hydroxyl groups. Succinate moieties are attached to the amino moieties through amide bonds to form cleavable linkers attached to the microarray. Oligomers may be synthesis in situ onto the cleavable linkers and subsequently cleaved using a cleaving base. The cleaved oligomers are recoverable and include oligonucleotides.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application claims the benefit of priority under 35 U.S.C. § 120 to U.S. patent application Ser. No. 11 / 229,757, filed Sep. 19, 2005, which is incorporated by reference herein, and to U.S. patent application Ser. No. 10 / 877,568, filed Jun. 25, 2004, which is incorporated by reference herein and of which claims the benefit of priority under 35 U.S.C. § 119 to U.S. Provisional Patent Application Ser. No. 60 / 482,929, filed Jun. 27, 2003.TECHNICAL FIELD OF THE INVENTION[0002]This invention provides a plurality of different of double stranded RNA compounds (dsRNA) and single stranded short inhibitory RNA compounds (siRNA) generated on microarrays having cleavable succinate linker moieties attached at known locations, wherein the compounds are synthesized in situ on the cleavable succinate linkers.BACKGROUND OF THE INVENTION[0003]Microarray preparation methods for synthetic oligomers such as oligonucleotides (oligos) include the following:...

AI Technical Summary

Benefits of technology

The approach enhances oligonucleotide density and synthesis efficiency on microarrays and provides a stable, long-lasting antiviral therapy that effectively inhibits viral replication and combats resistance by using pools of siRNA to target multiple viral mRNAs.

Problems solved by technology

In general, formation of an ester linkage to an organic hydroxyl on a solid surface using a succinate is relatively difficult and results in relatively low yield.

Additionally, the reaction conditions require a relatively long period of time at relatively high temperature.

Antisense techniques had problems with target sequence accessibility in the mRNA.

Antisense also had problems with nonspecific side effects in clinical trials, particularly when chemically modified synthetic antisense oligonucleotides with modified backbones were used.

For example, when phosphorothiate-oligonucleotides were used and were designed to confer nuclease resistance, there was unanticipated binding to cellular proteins.

This caused nonspecific cellular disruptive effects.

Another problem was one of mRNA cleavage fragments produced by treatment with antisense oligonucleotides.

Viruses cause important functional alterations of the invaded cells, which can result in cellular death.

Although effective in some patients, such agents have been shown frequently to result in only a transient response or to have significant toxicity.

Another important limitation of antiviral therapy is the emergence of resistant mutants.

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